Plant Physiol. (1985) 77, 978-9830032-0889/85/77/0978/06/$0 1.00/0

Anaerobic Stress in Germinating Castor Bean, EthanolMetabolism, and Effects on Subcellular Organelles

Received for publication October 1, 1984 and in revised form December 14, 1984

ROBERT P. DONALDSON*, PATRICIA SOOCHAN, AND ALEXANDER ZARASDepartment ofBiological Sciences, George Washington University, Washington, D.C. 20052

ABSTRACT

Endosperms from castor beans (Ricinus communis) germinated for 0to 6 days were exposed to anoxia for 0 to 15 hours. Ethanol, the onlyalcohol detected by gas chromatography in the tissue, accumulates to aconcentration of 15 millimolar during the first 2 to 4 hours of anoxia andsubsequently decreases. The absolute amount of ethanol varies from 10micromoles per 5-day endosperm after 4 hours anoxia to less than 1micromole in 2-day endosperm after 4 hours. Lactate content is 2micromoles or less per endosperm. Alcohol dehydrogenase and pyruvatedecarboxylase activities, which are localized in cytosolic fractions, arenot greatly affected by anoxia. The recoveries of the marker enzymesand protein in endoplasmic reticulum (ER) and mitochondrial fractionsdecrease during anoxia. After 15 hours, the recovery of NADPH cyto-chrome c reductase is 15% of that in controls, fumarase is 50%, andcatalase is 75%.

Glyoxysomes and ER are capable ofconverting ethanol to acetaidehydewhich was measured using the fluorogenic reagent, 5,5-dimethyl-1,3-cyclohexanedione. The glyoxysomal activity is dependent on a hydrogenperoxide-generating substrate and the ER is dependent on NADPH.However, these activities are less than 3% of the alcohol dehydrogenaseactivity.

Hypoxic stress and alcoholic fermentation are common fea-tures of plant metabolism. Ethanol is produced by flooded seed-lings (1, 18, 19), ice encased leaves (2), root nodules (20), andtissues exposed to S02 and 03 (10). The endosperm ofgerminat-ing castor bean endosperm is an appropriate model to studyanaerobic stress and alcohol metabolism. This tissue responds toanoxia by generating ethanol and smaller amounts of lactate(11). ER, mitochondria, and glyoxysomes, subcellular compo-nents which depend on 02 have been isolated and well charac-terized (3, 6, 21). The glyoxylate cycle has been shown toparticipate in ethanol metabolism (17). In the earlier investiga-tion of anoxic castor bean endosperm, Kobr and Beevers (1 1)followed ethanol accumulation for 2 h in endosperms after 5 dgermination. However, plant tissues are often capable ofproduc-ing ethanol for longer periods of time (1, 4).One purpose of this study was to determine the extent and

regulation ofethanol production in castor bean during prolongedanoxia as a function of seedling age. Also, we examined thepossibility that other alcohols might be produced and metabo-lized during anoxia. Because the endosperm initially containsvery little carbohydrate (7), it was expected that little or noethanol would be generated prior to the establishment of gluco-neogenesis. We explored the possibility that the capacity forethanol generation might be regulated by pyruvate decarboxylaseand alcohol dehydrogenase, the enzymes responsible for the final

steps of ethanol production.The second major aim of this study was to examine the

subcellular locations of ethanol metabolism and the effects ofanoxia on 02 requiring organelles. It has been reported that ratliver microsomal Cyt P450 and peroxisomal catalase are capableof oxidizing ethanol (14, 16). The significance of these activitiesrelative to the alcohol dehydrogenase is not clearly established.Thus, we examined the capacities of ER and glyoxysomes tometabolize ethanol in castor bean endosperm. We followed theeffects ofanoxia on the recoveries ofmarker enzymes and proteinin these subcellular fractions as well as mitochondria.

MATERIAILS AND METHODSCastor beans (Ricinus communis var Hale) were grown in

water-saturated vermiculite in the dark at 30C. The seeds werenot presoaked because of possible O2 limitations. The endo-sperms were harvested at the desired stage of germination, 1 to6 d. Endosperms were placed, without added liquid, in flasksconnected in series to a source ofnitrogen or air bubbled throughdistilled H20. The flasks were placed in a 30°C incubator. Ateach designated time, the last flask containing the appropriateamount of endosperm was removed such that the flow of gas tosubsequent samples was not interrupted during incubation.Gas Chromatographic Ethanol Measurement. Endosperms

from three seedlings were placed in an ice-cold mortar containing6 ml 50 mM Tricine (pH 7.5) and homogenized. The homogenatewas quickly transferred to a cold l0-ml screw-capped test tubeand centrifuged for 10 min at 1000g. The lipid layerwas removedand 3.2 ml of the supernatant was transferred to another cold10-ml tube. Protein precipitation was performed using a modi-fication of the procedure described by Cooper (5). The 3.2 ml ofsupernatant was vortexed with 0.15 ml of0.67 N H2SO4 and 0.15ml of 10% sodium tungstate and allowed to stand on ice for 5min. It was then centrifuged for 5 min at 1000g. One ml of thesupernatant was mixed with 1 ml of 2.65 mm isobutanol, theinternal standard, and 2 ,ul of this was injected in the gaschromatograph. The sample was analyzed on a Hewlett-Packard5840A gas chromatograph with a 5840A GC terminal, using aglass column (1.83 m x 2 mm) packed with Supelco Poropak Q,80/100 mesh. The oven temperature was 170°C, the helium flowwas 40 ml/min, and the flame ionization detector temperaturewas 250C. The number of moles of ethanol was determinedfrom the peak area relative to the internal standard, isobutanol.All analyses were performed in duplicate.

Lactate Measurement. Portions ofendosperm from three seed-lings were weighed to 0.5 g and homogenized in 4 ml of 8%HC104 with a mortar and pestle (9). The homogenate wascentrifuged at 1000g for 10 min, the lipid layer removed, andthe supernatant decanted. The reagents from a Sigma lactic acidkit were used in the analyses. To a l-ml cuvette were added 50Ml supernatant, 0.74 ml of the buffer-NAD mixture, 50 ,l of thelactate dehydrogenase suspension. The Aw0 was recorded before

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ETHANOL METABOLISM IN CASTOR BEAN ENDOSPERM

adding the dehydrogenase and after incubating for 40 min at37C. The amount of lactate was calculated from the change inabsorbance. The recovery of a known amount of lactate addedto the homogenate was 86%.

Cell Fractionation. The ER, mitochondria, and glyoxysomeswere separated from the cytosol by centrifuging the 270g super-natant from homogenized endosperm on a sucrose gradient in avertical rotor as described by Donaldson (8).Enzyme Assays. Alcohol dehydrogenase was measured in a 1-

ml reaction mixture containing 60 mm Tris (pH 9.2), 25 mMMgCl2, 0.5 mm NAD, 50 mm alcohol, and 20 jl of sample. Therate ofNADH formation was calculated from the change in A_wrecorded by a Perkin Elmer 555 spectrophotometer using anextinction coefficient of 6.22 mM-2 for the NADH. Routineassays, Km, determinations, and substrate specificity studies weredone using samples of 270g supernatants of endosperm homog-enates.

Pyruvatedecarboxylase was measured according to the methodof Leblova and Valik (13) using a Perkin-Elmer 240 fluorescencespectrophotometer. Samples were prepared by homogenizingendosperms from three seedlings with 6 ml 0.2 M sodium phos-phate (pH 6.0) in a mortar and pestle. After centrifugation at7000g for 10 min, the lipid layer was removed and the superna-tant decanted. The enzyme activity in a 0.2-ml sample of thesupematant was determined in a 2-ml reaction mixture contain-ing 60 mm sodium phosphate (pH 6.0), 20 mm sodium pyruvate,0.5 mm thiamine pyrophosphate, and 0.5 mM MgCI2. The mix-ture was incubated for 10 min at 40°C and then 2 ml of thedimedone reagent was added to the mixture and placed in aboiling water bath for 6 min. (The dimedone reagent consistedof 0.3 g 5,5-dimethyl-1,3-cyclohexanedione, 2.5 g ammoniumacetate, 0.4 ml acetic acid made up to 100 ml with water.) Theamount of acetaldehyde produced was determined by measuringthe fluorescence at 460 nm when excited at 396 nm. The 100%fluorescence was set with a standard consisting of 2.5 mm acet-aldehyde mixed with an equal volume of dimedone reagentdeveloped in a boiling water bath for 6 min. The amounts ofacetaldehyde produced were calculated from the linear regressionequation generated by a series of known acetaldehyde concentra-tions (up to 2.5 mM) included in the enzyme reaction mixturedescribed above. This compensated for quenching of fluores-cence by components of the reaction mixture. The sensitivityand repeatability of the dimedone measurement of acetaldehydewere later improved as described below.

Ethanol oxidation activities in ER and glyoxysomal fractionsfrom sucrose gradients were compared to cytosolic alcohol de-hydrogenase by measuring the acetaldehyde production usingthe dimedone reagent described above. The reaction mixture foralcohol dehydrogenase was as described above except that a 5-ml final volume contained 50 zA of the cytosol fraction. Thereaction mixture for ER contained 50 mm K-phosphate (pH 7.4),0.2 mm NADP, 3 mM glucose-6-P, 0.06 units glucose-6-P dehy-drogenase, 50 mM ethanol, and 0.1 to 1.0 ml ER sample in a 5-ml final volume. The glyoxysomal reaction mixture contained30 mm K-phosphate (pH 7.4), 0.15 mg/ml BSA, 0.01% TritonX-100, 0.1 mm CoA, 0.2 mM NAD, 10 FM palmitoyl CoA, 50mm ethanol, and 0.5 ml glyoxysomal fraction in a 5-ml finalvolume. The three different reaction mixtures were incubated at30C. At timed intervals, duplicate 0.5-ml samples were takenfrom the mixtures, mixed immediately with I ml dimedonereagent containing 0.5 ml H20, and developed in a boiling waterbath for 40 min. The time intervals for alcohol dehydrogenasewere 1, 3, 4, and 5 min. The intervals for ER and glyoxysomeswere 1, 4, 6, and 8 min. The fluorescence was compared toknown amounts of acetaldehyde, 0.5 to 100 nmoL developedwith dimedone reagent. Fluorescence was read at 345/460 nm.

length were found to yield higher and more stable fluorescencereadings than the conditions used previously (13).

Protein concentrations were determined by the LowIy method.Catalase (6), fumarase (6), and NADPH Cyt c reductase (21)were assayed as decibed. All reagents used in the enzyme assayswere obtained from Sigma.

RESULTS

Gas Chromatography. This method was used rather than anenzymic technique to allow detection of other substances, forexample other alcohols, which might accumulate during anaer-obic metabolism. Ethanol was the only product detected. Theminor components observed (Fig. 1) were present in the endo-sperm before anoxia and did not change in amount duringanoxiaThe recovery of ethanol was determined by adding a known

amount of ethanol to the homogenization of an endospermsample. The sample was prepared for GC as descnbed. Theamount of ethanol recovered was 80% of the amount addedindicating that our measurements underestimated the ethanolaccumulation by 20%.

and Lactate Production during Anoxia. When castor

ban endosperms are placed in a N2 atmosphere, ethanol andsmall amounts of lactate accumulate (Table I). Endosperms inlater stages of germination produce more ethanol but generateless lactate than endosperms at earlier stages. The amount ofaccumulated ethanol declines after a few hours of anoxia. Thedecline begins after 2 h in 2- and 34 endosperms, after 3 h in 4-

d tissue, and after 4 h in 54 tissue. This indicates that ethanolproduction is limited in some way related to seedling develop-ment.The concentration of ethanol relative to the weight of water

imbibed was estimated to be 15 mm in endosperms regardless ofseedling age.

Pyruvate Decarboxylase and Alcohol Dehydrogenase. Theseenzymes are responsible for converting the product of glycolysis,pyruvate, to ethanol. Also, alcohol dehydrogenase initiates thereutilization of alcohol when 02 is restored. Since ethanol pro-duction appears to be limited, these enzymes were measured atdifferent stages of germination. Pyruvate decarboxylase activityincreases during germination and is enhanced by anoxia (TableI). The alcohol dehydrogenase activity in castor bean is 230nmol/min per endosperm prior to germination, increases to 650nmol/min after I d and persists at 500 nmol/min from 2 to 5 d.As shown below, anoxia diminishes this activity slightly.The cytosolic alcohol dehydrogenase ofcastor bean endosperm

has the capacity to oxidize a variety of alcohols as do other plant

1.4

6.3

RETENTION TIME

FIG. 1. GCofdeproteinized homogenate ofendospermafterexposureto 2 h anoxia. The peak at 1.4 min is ethanol The peak at 6.3 min isisobutanol, the internal standard whih was added in known amounts tothe homogenate. The chromatography was performed on Poropak Q,

The 40-min development time and the 345-nm excitation wave-

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980 DONALDSON ET AL.

Table I. Accumulation ofEthanol and Lactate in Excised Endospermduring Exposure to Anoxia

The sample for each time point consisted of three endosperms in asingle flask. Each value represents an average of duplicate samples. Therange did not exceed 25% of the mean.

Accumulation of Ethanol and Lactate atTime of Following Times in Anoxia

Germination0 20 min 40 min I h 2 h 3 h 4h 5h

d nmol ethanol/endosperm2 0 0.1 0.3 0.6 1.8 0.9 0.7 0.83 0 0.3 1.1 1.8 2.5 2.3 1.7 1.54 0 0.4 1.2 1.9 3.2 5.2 4.8 3.65 0 0.4 2.1 2.8 6.5 8.9 10.0 6.1

nmol lactatelendosperm2 0.0 NDa ND 0.3 0.3 1.9 ND 1.13 0.5 ND ND 0.7 1.9 2.1 ND 2.34 1.5 ND ND 0.9 1.1 1.6 ND 1.95 0.2 ND ND 0.5 0.3 0.3 ND 0.2

a Not determined.

Table II. Pyruvate Decarboxylase in Germinating Castor BeanEndosperm

Castor beans were germinated from 2 to 5 d and the excised endo-sperms were exposed to anoxia for the time indicated. Pyruvate decar-boxylase activity was measured in endosperm homogenates using thedimedone reagent described in "Materials and Methods."

Activity of PyruvateTime of Decarboxylase at Following

Germination Hours in Anoxia0 3 5

d Mumol-endosperm-' h-'2 7.8 7.4 8.33 12.4 13.2 14.24 16.6 17.2 17.95 15.7 15.1 17.7

tissues (12). However, ethanol is the only alcohol detected incastor bean and is the preferred substrate. The dehydrogenaseactivity decreases with increasing chain length of the alcohol.Relative to ethanol the activities are as follows, propanol andbutanol 50%, pentanol 30%, hexanol 10%, and heptanol 3%.Also, the Km is proportional to carbon number, ranging from 10

mm for ethanol to 80 mm for pentanol. The greatest activity isobtained with 50 mm ethanol; higher concentrations are inhibi-tory.

Subcellular Distribution of Alcohol Metabolism. Figure 2shows that alcohol dehydrogenase and pyruvate decarboxylaseare located in the cytosolic fractions of germinating castor beanendosperm. These fractions contain other cytosolic componentssuch as most of the enzymes of reversed glycolysis and thepentose cycle as previously reported (8, 15). Other subcellularcomponents may contribute to ethanol oxidation. Both the ERand glyoxysomal fractions will convert ethanol to acetaldehydewhich can be detected fluorometrically using the dimedonereagent (Fig. 3). In each case, the spectral characteristics of theproduct are the same as acetaldehyde, maximum excitation at395 nm and maximum emission at 460 nm. The ER activity isdependent upon an NADPH-generating system and is inhibitedby CO (Table III), indicating that a Cyt P450 monooxygenaseis responsible (21). The glyoxysomal activity requires a H202source, such as fatty acid oxidation. The acetaldehyde producedin the absence of ethanol may be derived from acetate released

AIR

10 20 30

Plant Physiol. Vol. 77, 1985NITROGEN

10 20 30

VOLUME (ml)

FIG. 2. Subcellular fractionation of excised 4-d endosperm exposedto air or N2 for 15 h. After exposure, 15 g of endosperm from eachtreatment was homogenized and centrifuged at 270g, 10 min, to removethe fat, plastids, and nuclei. Then 13.5 ml ofthe supernatant was layeredon a 20 ml, 18% to 59% (w/w) linear sucrose gradient and centrifugedin a vertical rotor for 25 min at 25,000 rpm as described previously (8).The units are: protein, mg; alcohol dehydrogenase, 5umol/min; pyruvatedecarboxylase, umol/min * 10; catalase, mmol/min; fumarase, umol/min;NADPH Cyt c reductase, nmol/min.

from the fatty acid. Under the conditions used here, the glyoxy-somal oxidation of ethanol exhibits saturation kinetics and anapparent Km value of 20 mm for ethanol (Fig. 4) which is in thesame range as the cytosolic alcohol dehydrogenase. The kineticsofthe glyoxysomal activity are probably a function of the rate ofH202 production.

Total ethanol oxidation capacity was compared in subcellularfractions obtained from normal, aerobically germinated castorbean endosperms (Fig. 5). The cytosolic alcohol dehydrogenasehas the greatest capacity to convert ethanol to acetaldehyde. Theglyoxysomal system has a lower capacity and the ER has theleast.

Effect of Anoxia on Subcellular Fractions. The ability of thevarious subcellular components to oxidize ethanol followinganoxia and return to 02 requires that they survive anoxia.Subcellular fractions were isolated from endosperms which hadbeen germinating 4 d and then exposed to anoxia for up to 15h. These were compared to fractions obtained from endospermswhich were excised but maintained in air (Fig. 2). The proteinassociated with the ER fractions is diminished during anoxia to75% at 6 h and 60% at 12 h. Some of the protein in thesefractions is cytosolic and the per cent recovery of ER protein isoverestimated. The ER band is no longer visible in the centrifuge

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ETHANOL METABOLISM IN CASTOR BEAN ENDOSPERM

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350 400 450 500WAVELENGTH

FIG. 3. Fluorescence spectra of ethanol oxidation products after re-action with the dimedone reagent. Excitation: the emission or analyzerwavelength was held constant at 460 nm while the excitation wavelengthwas varied from 300 to 450 nm. Emission: the excitation wavelength washeld constant at 394 nm while the emission wavelength was varied from400 to 550 nm. The three pairs of peaks shown represent (A) theglyoxysomal palmitoyl CoA-dependent ethanol oxidation product, (B)the ER NADPH-dependent ethanol oxidation product, and (C) acetal-dehyde.

Table III. Conversion ofEthanol to Acetaldehyde by ER andGlyoxysomes

The complete reaction mixture was as described in "Materials andMethods." The ER fractions from a sucrose gradient were diluted with 2volumes of 50 mm Tricine buffer and centrifuged at 40,000 rpm for 30min in a Dupont A840 rotor. The pellet was resuspended in a volumeof 50 mm Tricine equal to the original volume ofthe combined fractions.The centrifugation and resuspension were repeated.

Rate of AcetaldehydeAssay Formation in

ConditionER Glyoxysomes

nmol. ml' . min-'Complete 13.5 560Omit subcellular fraction 0.0 113Omit ethanol 0.0 213Omit substrate' 0.0 0Omit cofactorsb 0.0 133Complete, CO treatedc 4.5 NDd

a Substrates: ER, glucose-6-P and glucose-6-P dehydrogen-ase. b Cofactors: ER, NADP; glyoxysomes, NAD and CoA. C COtreatment: CO gas was bubbled through the complete reaction mixturein the cuvette for 30 s. d Not determined.

1/ S (mmi1)

FIG. 4. Double reciprocal plot of palmitoyl CoA-dependent ethanoloxidation in glyoxysomes.

PERCENT OF TOTALETHANOL OXIDATION

CYTOSOLIC ALCOHOL DEHYDROGENASE

ETHANOL + NAD' -4 ACETALDEHYDE + NADH + H+ 96.9

ER CYTOCHROME P450

ETHANOL + NADPH + H+ + 02 - ACETALDEHYDE + NADPt+ 2H20

GLYOXYSOMAL FLAVIN OXIDASE AND CATALASE

ACYL CoA + 02 - ENOYL CoA + H20

ETHANOL + H202 - ACETALDEHYDE + 2H20

0.5

2.6

FIG. 5. Ethanol oxidation in cytosol, ER, and glyoxysomes. Fractionswere isolated from castor bean endosperm after 4 d germination. Thecytosolic fractions were combined, the ER fractions were combined, andthe glyoxysomal fractions were combined. The acetaldehyde productionrate was determined in each and reported as a percentage relative to thetotal activity in all three. The rate of acetaldehyde production from 50mm ethanol was measured in the appropriate reaction mixtures foralcohol dehydrogenase, NADPH mixed function oxidase, and palmitoylCoA oxidation, respectively (see "Materials and Methods"). Acetalde-hyde was measured using the dimedone reagent. No correction was madefor acetaldehyde produced without ethanol.

tube after 12 h. NADPH Cyt c reductase activity in the 25 to35% sucrose region of the gradient, where ER is usually found,is much lower after 9 h and is almost immeasurable after 12 h(Fig. 6). The recovery of protein in mitochondrial fractions is50% at 6 h and 18% at 12 h. The marker enzyme, fumarase, isalso diminished by anoxia. Alcohol dehydrogenase activity isdecreased slightly as are glyoxysomal protein and catalase.

DISCUSSION

This investigation shows that excised castor bean endospermaccumulates ethanol and smaller amounts of lactate duringanoxia. No other alcohols are detected by GC, although thealcohol dehydrogenase is capable of oxidizing C3 through C7alcohols. Endosperms in earlier stages ofgermination accumulateless total ethanol than the 5-d tissue. However, the ultimateconcentration achieved is about the same in all stages, 15 mm.The production of ethanol is sustained for 2 to 4 h and thendeclines.

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Plant Physiol. Vol. 77, 1985

9

HOURS ANOXIA

FIG. 6. Effects of anoxia on the recovery of marker enzymes insubcellular fractions from castor bean endosperm. The tissue was excisedafter 4 d germination, exposed to anoxia for the period of time indicated,and fractionated as shown in Figure 2. The marker enzyme activities inthe appropriate fractions were totaled and compared to the total activityin the corresponding fractions obtained from the unexposed endosperm.Cytosol fractions: alcohol dehydrogenase (0); glyoxysomal fractions:catalase (U); mitochondrial fractions: fumarase (0); ER fractions:NADPH Cyt c reductase (0).

There is a general correspondence of ethanol production ca-

pacity with increased activities of pyruvate decarboxylase andalcohol dehydrogenase during germination. However, neither ofthese enzymes is rate limiting in ethanol generation. Our meas-

urements and those previously reported ( 11) indicate that ethanolis generated at a rate of about 4 umol/h per endosperm in a 5-dcastor bean. Pyruvate decarboxylase activity at 5 d is 15 Mmolacetaldehyde/h per endosperm. Alcohol dehydrogenase has an

activity of 30 ,mol/h per endosperm, at least in the reversedirection. The decline in ethanol generation is not associatedwith a decrease in these activities during anoxia. Therefore, theprocess must be regulated elsewhere.The potential to accumulate larger amounts of ethanol is

correlated with the development of gluconeogenic activity andincreased carbohydrate content in castor bean. Seedlings whichcontain mostly stored carbohydrate such as rice and oats are

capable of sustaining ethanol production for longer periods oftime, 24 to 92 h, and achieve concentrations in the range of 40mM (1, 4). In castor bean, the amount of ethanol produced ismuch less than the amount of available carbohydrate. Desveauxand Kogane-Charles (7) observed that the greatest accumulationof sugar is detected in the endosperm at the point in time whenabout half of the fat has been degraded. After 6 d germination at25°C, they measured 260 umol sucrose per endosperm and 96,umol glucose. We detected only 10 ,mol ethanol per endospermat about the same stage of germination, 5 d at 30°C.The rate and extent of ethanol production are not directly

determined by pyruvate decarboxylase and alcohol dehydrogen-ase activities or by carbohydrate availability as discussed above.Glycolysis may be regulated by enzymes such as phosphofruc-tokinase and pyruvate kinase. The measurements made by Kobrand Beevers (I 1) of glycolytic intermediates in castor bean en-dosperm implicate these two enzymes in establishing the rate ofglycolysis at the onset of anoxia. Such regulation would not,

however, explain the eventual decline in ethanol during anoxia.Compartmentation of sugar, in the vacuole, could limit theextent of ethanol production.The second purpose of our study was to investigate the sub-

cellular location(s) of ethanol oxidation and the effects of anoxiaon subcellular organelles. Our studies show that ethanol oxida-tion may take place in several subcellular locations involvingthree different mechanisms. In the cytosol, the NAD-dependentalcohol dehydrogenase is responsible. The ER is capable ofNADPH-dependent oxidation of ethanol. Glyoxysomes can con-duct the peroxidation of ethanol when provided with a H202generating substrate such as fatty acid. The greatest capacity,however, seems to reside with the cytosolic dehydrogenase.We observed a diminished recovery of marker enzymes and

protein in ER and mitochondrial fractions after 6 to 15 h anoxia.This suggests that mitochondria and ER are degraded duringanoxia while the glyoxysomes survive to a greater extent. Thisincreases the possibility that the glyoxysomes could make asignificant contribution to ethanol metabolism during recoveryfrom anoxia.The rate of NADH generation by alcohol dehydrogenase may

exceed the capacity of mitochondrial oxidation especially duringthe initial stages of recovery when the ethanol concentration isrelatively high, 15 mm. Thus, the amount of free NAD availablecould limit cytosolic flux and increase the proportions of ethanolmetabolized in the glyoxysomes. Also, there are a variety ofH202-generating oxidases in glyoxysomes in addition to the acylCoA oxidase which was supplying H202 in our measurements.Thus, the total rate of peroxide production, and ethanol peroxi-dation, is potentially greater than we measured. In vivo measure-ments of catalase status in liver indicate that peroxisomes maybe responsible for up to 30% of the ethanol metabolized (16).

LITERATURE CITED

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2. ANDREWS CJ, MK POMEROY 1979 Toxicity of anaerobic metabolites accu-mulating in winter wheat seedlings during ice encasement. Plant Physiol 64:120-125

3. BEEVERS H 1978 The role of mitochondria in fatty seedlingtissues. InG Ducet,C Lance, eds, Plant Mitochondria. Elsevier/North-Holland Biomedical Press,New York, pp 365-372

4. BERTANI A, I BRAMBILLA, F MENEGUS 1980 Effect of anaerobiosis on riceseedlings: growth, metabolic rate, and fate of fermentation products. J ExpBot 31: 325-331

5. COOPER JDH 1971 Determination of blood ethanol by gas chromatography.Clin Chim Acta 33: 483-485

6. COOPER TG, H BEEVERS 1969 Mitochondria and glyoxysomes from castorbean endosperm, enzyme constituents and catalytic capacity. J Biol Chem244: 3507-3513

7. DESVEAUX R, M KOGANE-CHARLES 1952 Etude sur las germination desquelques graines oleagineuses. Ann Inst Natl Rech Agron 3: 385-416

8. DONALDSON RP 1982 Nicotinamide cofactors (NAD and NADP) in glyoxy-somes, mitochondria and plastids isolated from castor bean endosperm. ArchBiochem Biophys 215: 274-279

9. GUTMAN I, AW WAHLEFELD 1979 L-(+)-Lactate determination with lactatedehydrogenase and NAD. In HU Bergmeyer, ed, Methods of EnzymaticAnalysis, Ed 2, Vol 3. Academic Press, New York, pp 1464-1468

10. KIMMER TW, TT KOZLOWSKI 1982 Ethylene, ethane, acetaldehyde and ethanolproduction by plants under stress. Plant Physiol 69: 840-847

1 1. KOBR MJ, H BEEVERS 1971 Gluconeogenesis in the castor bean endosperm I.Changes in glycolytic intermediates. Plant Physiol 47: 48-52

12. LANGSTON PJ, C PACE, G HART 1980 Wheat alcohol dehydrogenase isozymes.Plant Physiol 65: 518-522

13. LEBLOVA S, J VALIK 1980 Partial charcterization of pyruvate decarboxylaseisolated from germinating pea seeds. Aust J Plant Physiol 7: 35-40

14. LIEBER CS, LM DECARLI, S MATSUZAKI, K OHNISHI, R TESCHKE 1978 Themicrosomal ethanol oxidizing system (MEOS). Methods Enzymol 52: 355-367

15. NISHIMURA M, H BEEVERS 1979 Subcellular distribution of gluconeogeneticenzymes in castor bean endosperm. Plant Physiol 64: 31-37

16. OSHINO N, D JAMIESON, T SUGANO, B CHANCE 1975 Optical measurement ofthe catalase-hydrogen peroxide intermediate (compound I) in the liver ofanaesthetized rats and its implication to hydrogen peroxide production in

situ. Biochem J 146: 67-77

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ETHANOL METABOLISM IN

17. PETERsoN CA, EA COSSINS 1966 Participation of the glyoxylate cycle in themetabolism of ethanol by castor bean endosperm tissue. Can J Biochem 44:423-432

18. POMERY MK, CJ ANDREWS 1979 Metabolic and ultrastructural changes asso-ciated with flooding at low temperature in winter wheat and barley. PlantPhysiol 64: 635-639

19. RUMPHO ME, RA KENNEDY 1981 Anaerobic metabolism in germinating seeds

CASTOR BEAN ENDOSPERM 983

of Echinochola cus-galli (barnyard grass). MetaboIite and enzyme studiesPlant Physiol 68: 165-168

20. TXmA S, TA LARUE 1982 Enzymes for acetaldehyde and ethanol formationin legume nodules. Plant Physiol 70: 388-392

21. YOUNG 0, H BEEvats 1976 Mixed function oxidases from germinating castorbean endosperm. Phytochemistry 15: 379-385

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